The retina is not a static tissue, but a dynamic population of cells whose function is modulated through the day. The circadian regulation of the retina is evident from the level of gene transcription to the connectivity of cell networks. The circadian regulation of gene transcription includes genes involved in cellular metabolism and synaptic transduction. This daily rhythmicity of retinal physiology persists in the absence of light and in many cases even when the retina is cultured in isolation. This demonstrates that the circadian rhythms of the retina are controlled by local circadian clocks. Intriguingly, these retinal circadian clocks are also able to synchronize, or entrain, to light:dark cycles when cultured in a dish. These are the only known mammalian circadian oscillators which exhibit this light sensitivity. The identity of the cell type or types which exhibits robust circadian gene expression and susceptibility to light cycles is still unknown. Using a bioluminescent reporter of the circadian clock gene, Per2, the phase, amplitude and period of the circadian clocks present in many mouse tissues can be measured in organotypic tissue culture as a representation of the animal's endogenous rhythms prior to culture of the tissue. This technique has confirmed that the cells in the mouse retina which express the circadian clock are viable in culture and offers a unique opportunity to determine their identity. Using immunohistochemistry, the retinal cells which are strongly expressing PER2 as synchronized by a light:dark cycle will be identified by co-labeling with markers for retinal cell types. Also, mutations in genes which reduce or remove the function of key retinal cell types will be tested for their influence on retinal circadian rhythms. Because the cultures are still light entrainable, this also offers an opportunity to determine the wavelength of light to which the rhythms are most sensitive, which provides insight into the photoreceptive molecule. In addition, tissue culture allows for the use of pharmacology to test the cellular pathways involved in photo transduction. Inhibitors of known visual second messenger pathways and other circadian regulatory pathways will be used in the presence of light:dark cycles to test their influence on synchronization to light. This will be done with complementary genetic mutations of known retinal cell types as described above. Finally, the ontogeny of retinal circadian clocks and their light sensitivity will be assessed in mouse pups in order to describe the onset of circadian rhythmicity in the developing retina. These experiments will provide answers to fundamental questions in regular retinal anatomy and physiology. They will also open up future avenues of research and potential therapeutic regulation of retinal function.

Public Health Relevance

The sensitivity of human vision and the electrical response of the retina to light exhibit a daily fluctuation as a circadian rhythm. The retina contains cellular clocks which maintain these local circadian rhythms, however the cellular identities are not known. This proposal seeks to identify these retinal clock cells and the molecular pathway by which they are synchronized by light:dark cycles.

National Institute of Health (NIH)
National Eye Institute (NEI)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F03A-N (20))
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Agarwal, Neeraj
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University of Washington
Schools of Medicine
United States
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Buhr, Ethan D; Van Gelder, Russell N (2014) Local photic entrainment of the retinal circadian oscillator in the absence of rods, cones, and melanopsin. Proc Natl Acad Sci U S A 111:8625-30